The long term goal of this proposal is to gain an understanding of the mechanisms by which genome RNA of the negative strand virus group is transcribed and replicated. This group comprises some of the most serious human pathogens in terms of both mortality and morbidity, such as rabies, influenza, mumps, etc. Vesicular stomatitis virus (VSV) - a prototype of negative stranded rhabdoviruses, provides an excellent model system to study transcription and replication in vitro. An RNA-dependent RNA polymerase (L and NS protein) is packaged within the virion which faithfully copies the genome RNA in vitro and in vivo; this enzyme complex, in association with the N protein is also involved in the replication process. Thus, a thorough study of the structure and function of the L and NS proteins, as well as the nucleocapsid protein (N), will be essential to understand the precise mechanism of transcription and replication of VSV. This research proposal is designed to use clonaly expressed NS, N, and L proteins using SP6 transcription system and, by subsequent genetic manipulation of the genes, to study the functions of the domains within the polypeptides in the transcription and replication processes. This approach will lead to a better understanding of the roles of these polypeptides in viral gene expression and the eventual development of possible antiviral drugs. The specific aims of the grant are as follows: (1) Elucidate the functional role of the domains with the NS protein involved in binding to the template and the L protein, phoshorylation, and RNA chain elongation. In addition, determine the domains that govern specificity of interaction between the NS protein, the L protein and the N-RNA template using NS proteins of different serotypes; (2) Determine the functional role of various domains of the N protein involved in binding to the NS protein, and assembly with the genome RNA and replication in vitro; (3) Synthesize biologically active L protein from a cDNA clone and determine the domains involved in transcription. In addition, identify the domains involved in binding to nucleotides, protein kinase and capping activities.